MYCOTOXINS NATURAL vs SYNTHETIC CHEMICALS

• Are natural chemicals safer than synthetic chemicals? • Which set pose a greater risk? • Which set is more amenable to control and regulation? • Fundamental concept of Toxicology: all substances can be toxic it is a matter of DOSE

Mycotoxin contamination is a major food safety challenge . Mycotoxins are toxic compounds, harmful to human and animal health, that are produced by some fungi . Mycotoxins are products of metabolism of toxigenic fungi . Aflatoxins receive priority attention due to: – high pre- and postharvest contamination potential – widespread occurrence in diverse foods and animal feeds – extreme toxicological significance to humans and animals, – impacts on food safety, nutrition, public health, and markets and income Many fungi in one place!

Cladosporium Fusarium

Gibberella Fusarium

Penicillium Trichoderma

page 5 Basics: Agriculturally Important Mycotoxins • Fungi contaminate grains, food, and feed worldwide

• Mycotoxins are chemical compounds Gibberella Ear Rot caused produced by some fungi by Gibberella zeae • More than 400 mycotoxins identified worldwide, 30 concerning for human or animal health • 5 principle mycotoxins affect cereal grains (corn, wheat, rye, barley, oats) • aflatoxins, fumonisins, deoxynivalenol (vomitoxin), zearalenone, and ochratoxin A Wheat Scab Developing Fungus is Dependent on the Environmental Conditions During Pollination and Early Grain Development

Favorable Primary Mycotoxin(s) Fungi Conditions Grains Aspergillus flavus Hot and dry, Corn, Durum Aflatoxins Aspergillus parasiticus drought (in EU) Deoxynivalenol Corn, Wheat, Fusarium graminearum Cool, wet, humid at (Vomitoxin) Oats, Rye, Fusarium culmorum grain fill Zearalenone Barley, Durum Fusarium verticillioides Warm to hot, dry at Fumonisins Corn Fusarium proliferatum and after flowering Penicillium verrucosum Harvest conditions Ochratoxin A Corn determine

Basics: Mycotoxins in Grain and Feed

• Environmental stresses increase susceptibility • Planting, harvest, and storage practices also contribute to mycotoxin risk

• Most mycotoxins remain intact after cooking, drying, freezing or storage conditions. • Mycotoxins at very low concentrations (ppm or ppb) are capable of causing serious health problems for humans as well as animals. • There is usually no treatment for mycotoxin poisoning (mycotoxicosis). ppm: parts per million ppb: parts per billion Mycotoxin testing procedure

Procedural component Output

1) Sampling  Laboratory Sample • Sample size reduction (if necessary)

2) Sample Preparation  Analysis Sample • Grinding • Mixing An example of a • Subdividing grinder that would be used to grind a laboratory sample. 3) Analysis  Mycotoxin Result Mycotoxin Management Plan

• Most mycotoxin contamination happens in the field • Prevent contaminated grain from entering the facility • Pre-harvest preventative controls • Scouting and pre-harvest observations • Climate and weather conditions • At-receiving mycotoxin testing procedure What are Aflatoxins?

• Toxins produced by the fungi Aspergillus flavus and Aspergillus parasiticus • Aflatoxins are secondary fungal metabolites. • Aflatoxin types include B1, B2, G1, G2. • B1 is most prevalent and toxic aflatoxin.

Microscopic view : spore formation of Aspergillus Chemical structure of aflatoxin B1 • Detection: • Fluorescence can be used to detect presence of Aspergillus on crops • Biomarkers are used to detect aflatoxin exposures in humans

Aflatoxins • Aflatoxins cause health problems around the world including areas as diverse as Africa, Southeast Asia, Western Pacific, East Mediterranean and Latin America where as many as 5 billion persons may be exposed • Aflatoxins have a negative economic impact on agriculture through reduced marketing options for crops and adverse health effects on livestock • Aflatoxins are carcinogenic substances and may be present in a large number of foods. This toxin can cause cancer, cirrhosis of the liver. Aflatoxin distribution

• Exposure mainly from: – A. flavus: global distribution, produces B

classes of aflatoxins – A. parasiticus: Africa and the Americas,

produces B and G classes of aflatoxins

Partial list of foods: • Cereals – maize, sorghum, millet, rice, wheat • Oil seeds – groundnut, soybean, sunflower, cotton • Tree nuts – pistachio, almond, walnut, coconut • Spices – paprika, chile, black pepper, coriander, turmeric, ginger • Figs • Milk, cheese, meat, eggs Where?

• Aflatoxins spread via colonization and contamination of crops such as maize, wheat, rice and cotton.

• May contaminate vertebrate milk

• Foods found in: – Peanuts and various other nuts – Corn – Cottonseed – Stored Food Products

Pre-harvest risk factors: – High temperatures – Chronic drought – Heavy rains – Crop insect damage – Poor fertility – Weed competition – High crop densities Post-harvest risk factors: –High temperatures –Humidity Liver function

• One of the largest internal organs • Produces bile used to digest food • Metabolizes carbohydrates and lipids • Stores glycogen (for energy), key nutrients • Breaks down toxic substances Cytochrome P450 oxidase: • Found in high densities in liver • Oxidative enzymes that modify and degrade toxins • Absorb light at 450 nm http://www.biochem.ucl.ac.uk/bsm/proLig/pdbEntries/1pha/ Examples of Aflatoxin-Related Events

• United Kingdom – 1960s: Turkey X disease and bird die-offs attributed to ‘syndrome X’ – Both incidents were the result of acute aflatoxicosis • United States – 1998: Crop contamination • Aflatoxin contamination of maize (corn) in the south- eastern U.S. led to rejection rates of corn of up to 50%. • Aflatoxin contamination reached 1500 ppb (5 times the 300 ppb highest acceptable limit in animal feed set by the U.S. Food and Drug Administration) – 2006-2007: Crop contamination • Drought conditions and moisture stress led to aflatoxin on corn in Missouri which caused rejection of some harvested corn by buyers • Kenya – 2004-2005: Acute Aflatoxicosis and human mortality • Aflatoxin contamination of maize caused over 150 human deaths

Aflatoxins: Human, Animal, and Environmental Interactions Environment: extreme Fungal drought, growth / moisture, heat, aflatoxin compromised production plants

Contamination of human & animal food

Human consumption Breast milk In utero Milk Animal eggs consumption

Infants

Aflatoxins

• The major aflatoxins of concern are B1, B2, G1, and G2  usually found together in various proportions. Aflatoxin B is usually predominant, and it is the most toxic and carcinogenic. • The aflatoxins B1, B2 fluoresce blue and G1, G2 fluoresce green when viewed under a microscope. • A major metabolic product of aflatoxin B1 is aflatoxin M (usually excreted in the milk of dairy cattle that have consumed aflatoxin-contaminated feed).

4 major aflatoxins

2 metabolic M2 products AFLATOXIN TOXICITY

TOXICITY LD50 Aflatoxin B1 in different animals

SPECIE B1 DL50 mg/Kg os Anatroccolo 0,35 Tacchinotti 0,45 Pulcino 1-1,5 Suini 0,62 Cane 1-1,5 Pecora 2 Ratto 5,5-18 TOXICITY

• B1: high toxicity. Present in group 1 of IARC classification. Genotoxic and hepatocarcinogen. • by cytochrome P450 (P450) enzymes: activated to epoxide. • L’AFM1, metabolite of AFB1, shows an acute toxicity similar to those of AFB1, and chronic of 2- 10% Absorption

• Ingestion is the main route into the body • Gastrointestinal system will the be the first site of absorption

• After ingestion, Aflatoxins are absorbed by the blood stream and move through the fluid compartments • Enters Intracellular Fluid Compartment to disrupt DNA and Protein Synthesis

Aflatoxin→→→Epoxide ↑ P450

• Once converted, aflatoxins react with nucleic acids • They react with guanine in DNA and RNA leading to depurination • Aflatoxins can inhibit protein and DNA synthesis • DNA damage can also be mutagenic

Williams, J.H., T.D Phillips, P.E. Jolly, J.K Stiles and D. Agga. 2004. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am. J. Clin. Nutr.Nov;80(5):1106-1122. Mechanism of toxicologic damage

Also called steatosis, fatty liver can be a temporary or long-term condition, which is not harmful itself, but may indicate some other type of problem. Left untreated, it can contribute to other illnesses. It is usually reversible once the cause of the problem is diagnosed and corrected. The liver is the organ responsible for changing fats eaten in the diet to types of fat that can be stored and used by the body. Triglycerides are one of the forms of fat stored by the body and used for energy and new cell formation. The break down of fats in the liver can be disrupted by alcoholism, malnutrition, pregnancy, or poisoning. In fatty liver, large droplets of fat, containing mostly triglycerides, collect within cells of the liver. The condition is generally not painful and may go unnoticed for a long period of time. In severe cases, the liver can increase to over three times its normal size and may be painful and tender

29 Excretion

• Urine - The main pathway for excretion

• Milk

Hepatic necrosis • Feces Tremorgenic Nephrotoxicity Ascites- peritoneal cavity fluid Hemorrhages of Lungs and Brain (Cerebral Edema and Death) Carcinogenic Toxicology

• Acute aflatoxicosis – high dosage over short time – hemorrhage – acute liver damage – edema – altered digestion, absorption, and metabolism – death

Toxicology

• Chronic aflatoxicosis – impaired food conversion – slower growth – immunity problems – cirrhosis – liver cancer Mycotoxin testing procedure

Procedural component Output

1) Sampling  Laboratory Sample • Sample size reduction (if necessary)

2) Sample Preparation  Analysis Sample • Grinding • Mixing An example of a • Subdividing grinder that would be used to grind a laboratory sample. 3) Analysis  Mycotoxin Result Detection Methods Mycotoxin Regulations Concerning mycotoxins in food • Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs (consolidated version 2010-07-01) • Commission Directive 2008/128/EC laying down specific purity criteria concerning colours for use in foodstuffs • Regulation (EC) No 401/2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs (consolidated version 2010-03-13) • Commission Recommendation 2013/165/EU on the presence of T-2 and HT-2 toxin in cereals and cereal products Concerning mycotoxins in food of animal origin Commission Decision 2002/657/EC on implemention Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (consolidated version 2004-01-10) Concerning mycotoxins in feed • Directive 2002/32/EC on undesirable substances in animal feed (consolidated version 2010-03-02) • Commission Recommendation 2006/576/EC on the presence of deoxynivalenol, zearalenone, ochratoxin A, T-2 and HT-2 and fumonisins in products intended for animal feeding • Commission Recommendation 2013/165/EU on the presence of T-2 and HT-2 toxin in cereals and cereal products Recommendations on the prevention of mycotoxins Recommendation 2003/598/EC: prevention and reduction of patulin contamination in apple juice and apple juice ingredients in other beverages Recommendation 2006/583/EC: prevention and reduction of Fusarium-toxins in cerels and cereal products Recommendation 2012/154/EU: on the monitoring of the presence of ergot alkaloids in feed and food

Cl Ochratoxin A

N O

O OH O HO O Group 2B: "Possibly carcinogenic to humans" There is some evidence that it can cause cancer in humans but at present it is far from conclusive

There is inadequate evidence in humans for the carcinogenicity of ochratoxin A There is sufficient evidence in experimental animals for the carcinogenicity of ochratoxin A Toxicological effects:

target the renal proximal tubule

- Disrupt protein synthesis -Bind strongly to protein (albumin) -Interfere with synthesis of tRNA & mRNA -Disrupt carbohydrate metabolism -Increase the generation of free radical Formation of free radicals has been considered as one of the mechanisms for the carcinogenic/toxic effects of OA Ochratoxin A Symptoms of Exposure

• Ochratoxin A is primarily a kidney toxin • Swine and poultry are the primary livestock affected • Adverse effects in swine and poultry • Reduced feed intake, dehydration • Growth retardation • Kidney dysfunction • Diarrhea and excessive urine production • Reduced egg production (poults) • Vomiting (swine)

page 42 Aspergillus and Penicillium species producers of OTA in foodstuffs

(Ostry et al., Toxins 2013) The occurrence and amount of OTA in foodstuffs (Czech Republic, 2011-2013)

(Ostry et al., Toxins 2013) Mycotoxicoses: target organs

Mycotoxin Target

Aflatoxins Liver

Ochratoxins Kidney

Trichothecenes Mucosae

Peripheral Ergot alkaloids vascular system

Zearalenone Genitourinary tract µg Kg-1, µg L-1 Fusarium Toxins

Kingdom: Fungi Phylum: Ascomycota Genus: Fusarium Species: Many

Fumonisins Trichothecenes (T-2 toxin; DON) Zearalenone • These toxins are produced by fungi that grow mainly on corn, wheat, oats, and other cereals. • The levels of toxin produced are influenced by environmental factors – High levels produced when • hot dry weather which is fallowed by humidity • Plants are damaged by insects/birds • Most are heat resistant – Even if grains are cooked, mycotoxins still present (Fink- Gremmels, 1989).

General: Environmental role

• While these mycotoxins are harmful to mammals, they actually improve root growth of crops like corn. • They enable crop plants to survive through dry conditions and other stresses (Lee 2000).

Trichothecenes

• TCTCs are generally classified as macrocyclic (Type C) or nonmacrocyclic (Types A and B). Although more than 100 TCTCs have been identified, only a few frequently found in foods and feeds are potentially hazardous to human and animal health. • T-2 toxin, a highly toxic type A TCTC, is produced by F. tricinctum, F. sporotrichioides (major), F. poae, etc. • Unlike most mycotoxins, which are usually synthesized near 25C, the optimal temperature for T-2 toxin production is around 15C. • Almost all the major TCTCs, including T-2 toxin, are cytotoxic and cause hemorrhage, edema, and necrosis of skin tissues. • Desoxynivalenol (DON) is one of the most common mycotoxins found on animal feed

Trichothecenes

• TCTCs are generally classified as macrocyclic (Type C) or nonmacrocyclic (Types A and B). Although more than 100 TCTCs have been identified, only a few frequently found in foods and feeds are potentially hazardous to human and animal health. • T-2 toxin, a highly toxic type A TCTC, is produced by F. tricinctum, F. sporotrichioides (major), F. poae, etc. • Unlike most mycotoxins, which are usually synthesized near 25C, the optimal temperature for T-2 toxin production is around 15C. • Almost all the major TCTCs, including T-2 toxin, are cytotoxic and cause hemorrhage, edema, and necrosis of skin tissues.

Trichothecenes

• Desoxynivalenol (DON) is one of the most common mycotoxins found on animal feed • Zearalenone binds to estrogen receptors and leads to disturbances in fertility • High levels in farm animals causes serve morphological and function disorders of reproductive organs Vulvar prolapse

• May lead to pre-puberty in humans

Trichothecenes

• Mechanism of action – Inhibits protein synthesis by chemical inactivation of ribosomes – Acts like an estrogen and inhibits division of rapidly growing cells (i.e. intestine lining and bone marrow) – Inhibits/weakens effect of blood clotting Trichothecenes • The TCTC mycotoxicoses affect the gastrointestinal tract, hematopoietic, nervous, immune, hepatobiliary, and cardiovascular systems. • Effects of poisoning – Vomiting, Diarrhea, food rejection, GI inflammation, tissue necroses and impairment of: nerves, heart, lymph system, testes, and thymus – immunodepression • LD50: 10.5mg/kg in mice Zearalenone

• Estrogenic activity in swine and dairy that manifests as reproductive effects • Negative effects on cattle: • Infertility • Reduced milk production • Hyper-estrogenism • Negative effects on swine: • Enlarge mammae • Swelling of uterus and vulva • Atrophy of the ovaries • Withered testes • There are no FDA action, advisory, or guidance levels Fumonisin B1 & B2

• FB2 is mainly produced by F. verticilloides and F. proliferatum • Found mostly in maize-based human food and animal feeds • Most common fungus associated with corn intended for human consumption • Recently shown to be produced by Aspergillus niger in Thai coffee beans • Natural occurrence in corn found all over the world, from Argentina to Egypt to Nepal

Fumonisin B1 and B2

Fumonisin produced by Fusarium Maize in West Africa containing aflatoxin, dexoxynivalenol and fumonisin Fumonisin B1 & B2

• FB1 Most prevalent Fusarium toxin produced • Mechanism shared by FB1 and FB2 – Inhibits sphingosine biosynthesis • Sphingolipids are lipids found in neural tissue – Hepatoxic – Nephrotoxic – Suspected to be carcinogenic in humans Fumonisin B1 & B2

•FB1 Most prevalent Fusarium toxin produced •Mechanism shared by FB1 and FB2 Inhibits sphingosine biosynthesis (Sphingolipids are lipids found in neural tissue)

SFINGAMINA E SFINGOSINA, (sfingolipidi semplici)

Blocco

SFINGOMIELINA e CERAMIDI GANGLIOSIDI (sfingolipidi complessi)

Fumonisin Symptoms of Exposure

• Adverse effects in animals • Horses: Leukoencephalomalacia • Swine: Liver damage, pulmonary edema • Cattle and Sheep: Mild liver damage, moderate feed refusal

Infected kernels scattered or clustered are typical of Fusarium ear rot Source: Photo Courtesy of Pioneer HiBred Intl, Inc. Toxic phenolic substances

• Contribute to the bitter taste, flavor, color • Phenolic acids: , lignin, gallic acid, tannins • Highly toxic phenolic substances: coumarin, safrole, phenolic amines (gossypol, catecholamines), myristicin

Phenolics are a chemically diverse group: OH many different properties and functions. flavone

isoflavanone Biosynthesis of phenolics Shikimic acid pathway is most common in plants. Converts simple carbohydrates into aromatic amino acids. Not present in animals. Major types of phenolics 1. Simple phenolics - e.g. coumarins

2. Lignin - 2nd most abundant compound in plants

3. - two aromatic rings, 2 pathways anthocyanins, /

4. Condensed tannins polymerized flavonoids

5. Hydrolyzable tannins made of phenolic acids and sugars smaller molecules than condensed tannins

Flavonoids

• Plant pigmen that are widely present in human food (most are present as b-glucosides) More than 1 g ingested daily in the diets Divided into 6 groups - Flavanone - Flavone - - - Chalcone - Aurone

• often yellow (flavus Latin – yellow)

• known for a long time

• interest in them for – anti-inflammatory (and analgesic) properties – anti-allergic effects – antithrombotic, vasoprotective properties • decrease capillary fragility • phlebitis – changes in vessel walls in extremities -> plasma leakage -> oedema – mainly due to high oestrogen, sometimes in males – tumour inhibition promotion – protective for gastric mucosa Flavonoids - basic structure is two aromatic rings joined by a 3C bridge.

Fig. 13.10

a) anthocyanins b) flavones c) flavonols d) Source of flavonoids

• Sources of flavonoids include: apples, apricots, blueberries, raspberries, strawberries pears, black beans, cabbage, onions, and tomatoes.

• Fruit peel - oily orange peel : 2 mg nobitelin/100 ml oil 0.3 mg tangeretin/100 ml oil Toxicity of flavonoids

• In very high amounts (for example, 140 grams per day), flavonoids do not appear to cause unwanted side effects

• when raised to the level of 10% of total caloric intake, flavonoid supplementation has been shown non-toxic.

• Poor intake of fruits and vegetables - or routine intake of high-processed fruits and vegetables - are common contributing factors to flavonoid deficiency

• Toxicity: carcinogenic ( in cereal crops)

• At low concentrations  The effects of flavonoids are thought to be potentially anticarcinogenic because flavonoids can block and inhibit the excessive cell division characterized by cancer. Certain flavonoids can inhibit enzymes, such as protein kinases, that are involved in cellular proliferation and tumor progression. This is one reason flavonoids can be considered anticarcinogens.

• At high concentrationUniversity of California Berkeley (UC Berkeley) scientists led by C.F. Skibola and M.T. Smith  high concentrations of flavonoids may promote cancer formation  can damage the chromosomes and DNA in cells, leaving them more susceptible to cancer.

• can inhibit a number of enzymes that can alter normal body functions. • can involve genotoxic/carcinogenic effects or interaction with thyroid hormone biosynthesis. • can interfere with the metabolism of drugs and with mineral absorption in our bodies. • could inhibit the absorption of some nutrients, interact with certain drug pharmacokinetics and affect neurobehavioral development. • Daily intake: 150-250 mg/day • The FDA has not yet established recommended daily intake levels for flavonoids

“Just because something comes from a natural source doesn’t mean it can’t hurt you “

Tannins • Two types of tannins (polyhydric phenols) can be distinguished on the basis of degradation behavior and botanical distribution, namely hydrolyzable tannins and condensed tannins. • The hydrolyzable tannins are tannic acid, also known as gallotannic acid, gallotannin, or simply tannin. More easily hydrolyzed and degraded • The condensed tannins are formed by polymerization of flavonoids • Toxicity: cause acute liver injury, i.e., • liver necrosis and fatty liver. TANNINS (Basic structure) Hydrolysable Tannins Condensed Tannins

Glucose o oH oH oH oH oH o oH o oH oH oH o o o o o oH o Glucose OH

oH oH oH OH OH Gallotannins Ellagitannins Condensed tannins are polymerized flavonoids. Hydrolyzable tannins are made of phenolics and sugars.

The term “tannin” is derived from the tanning process in which raw animal hides are preserved by rubbing tannins on them. The tannins help to complex the proteins and keep them from degrading. Source of Tannins

• Fruits, tea (highest content), coffee, cocoa, grape, wine, grapes, grape juice • Tannins occur in many tropical fruits, including mango, dates, and persimmons. • A cup of ground coffee: 72-104 mg instant coffee: 11-128 mg • 1 g ingest of tannins per day

The contribution of the tannins in tea, coffee, and cocoa to the total tannin intake by humans is of particular importance.

Tea has the highest tannin content. Tannins can reduce nutritional value of tissues by binding to proteins, making them less digestible.

Fig. 13.16

Care for a spot of milk in your tea? Toxicity • If ingested in excessive quantities inhibit the absorption of minerals such as iron  lead to anemia

• This is because tannins are metal ion chelators,

• Tannins have been shown to precipitate proteins which inhibits in some ruminant animals the absorption of nutrients from high-tannin grains such as sorghum.

• Tannic acid does not affect absorption of other trace minerals such as zinc, copper, and manganese in rats

• In sensitive individuals, a large intake of tannins may cause bowel irritation, kidney irritation, liver damage, irritation of the stomach and gastrointestinal pain . CONCLUSION

• Tannins & its monomers have profound effects on health.

• Not advisable to take a large quantity of tannins, as they may be toxic.

• It is important to determine the correct dosage of tannins for promoting optimal health.